Drug delivery composition and method of fabrication

ABSTRACT

The methods of manufacture of a drug delivery composition. In some aspects, the methods include providing an organic phase, a biologically active ingredient, and an aqueous phase with a desirable pH (e.g., a pH at which the active ingredient has increased solubility in the aqueous phase compared to at neutral pH). After mixing of one or more of the aforementioned components, the resultant mixture is processed to provide the desired drug delivery composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/608,034, filed on Oct. 24, 2019 (published as US 20200054562), whichis the U.S. National Stage of International Patent Application No.PCT/US2018/031905, filed on May 9, 2018, which claims the benefit ofU.S. Provisional Patent Application No. 62/503,383, filed on May 9,2017, the contents of each of which are incorporated herein by referencein their entireties.

TECHNICAL FIELD

The disclosure relates to drug delivery compositions and methods offabrication, and more particularly to drug delivery compositions andmethods employing nanoparticles.

BACKGROUND

One of the key challenges to treating diseases, such as neoplasticdiseases, is exposing the targeted tissue to a sufficiently high drugconcentration. Numerous novel drug delivery strategies have beendeveloped with the rapid advances of nanotechnology. For example, anano-sized drug delivery system can address some of the knowndisadvantages of drugs, such as low bioavailability, poor solubility,and high cytotoxic side effects. Those of skill in the art have beenattracted to the use of polymeric nanoparticles to deliver therapeuticsfor myriad reasons, including controlling drug release, structuraldesign for targeting, and a functional design for delivery. However, asignificant disadvantage of using nanoparticles for drug delivery is themanufacturing process often leads to relatively low yields and poorloading efficiency.

Histone deacetylases (HDACs) are known to be key enzymes in cancerdevelopment and progression through their modulation of chromatinstructure and the expression and post-translational modification ofnumerous proteins. Aggressive dedifferentiated tumors, likeglioblastoma, frequently overexpress HDACs, while HDAC inhibition canlead to cell cycle arrest, promote cellular differentiation, and induceapoptosis. Although multiple HDAC inhibitors, such as quisinostat, areof interest in oncology due to their potent in vitro efficacy, theirfailure in the clinic as monotherapies against solid tumors has beenattributed to poor delivery. For example, some investigators report thatthe use of conventional nanoparticle-manufacturing processes results inonly 1-2% drug loading of some histone deacetylase inhibitors, such asquisinostat.

With that difficulty in mind, the inventors of the systems and methodsof drug delivery described herein sought to create new processes fordrug delivery systems, compositions, and methodologies. The inventorswere motivated to improve HDACi such as quisinostat loading ontopolymeric nanoparticles (NPs) such as poly(D, L-lactide)-b-methoxypoly(ethylene glycol) NPs.

SUMMARY

The invention herein is directed to therapeutic nanoparticles, themanufacture thereof, and use in treatment of a subject. In one exemplaryembodiment, the method of manufacturing therapeutic nanoparticles,comprises: mixing an organic phase with an aqueous phase to form amixture, wherein the organic phase comprises an organic solvent and ananoparticle comprising an amphiphilic polymer; adding a water insolublebiologically active ingredient, the active ingredient comprising anionizable group and having a partition coefficient of log P>0, whereinthe active ingredient is at least partially ionized in the aqueousphase, e.g., the active ingredient can be a weak acid; and removing theorganic solvent from the mixture. In a particular embodiment, the activeingredient is at least 70%, 80%, 90% or 99% ionized in the aqueous phaseand the active ingredient and the nanoparticle electrostaticallyinteract.

The invention also encompasses a method of fabricating therapeuticnanoparticles by preparing an aqueous phase; adjusting the pH of theaqueous phase; mixing an organic phase containing a nanoparticlecomprising an amphiphilic polymer with the aqueous phase; adding awater-insoluble biologically active ingredient, the active ingredientcomprising an ionizable group; and removing the organic solvent; whereinthe active ingredient has a higher water solubility in the adjusted pHthan in a neutral pH.

The active ingredient can be added at different stages of the method,for example, after the organic solvent is partially removed. Inexemplary embodiment, the method comprises dissolving the activeingredient in a solvent, e.g., dimethyl sulfoxide (DMSO), acetonitrile,or acetone.

In a specific exemplary embodiment, the method includes the steps of:(i) forming an organic phase comprising a polymer, such as anamphiphilic, hydrophobic, and/or hydrophilic polymer and an organicsolvent; (ii) adding an active ingredient to the organic phase; (iii)forming an aqueous phase comprising a hydrophilic solvent, wherein theaqueous phase further comprise surfactant and/or a stabilizing agent;(iv) mixing together the organic phase and a first portion of theaqueous phase to form an emulsification mixture; (v) emulsifying theemulsification mixture; (vi) adjusting a pH of a second portion of theaqueous phase to a desired pH that improves solubility of the activeingredient; and/or (vii) mixing together the emulsification mixture withthe second portion of the aqueous phase. In some embodiments, the methodalso includes evaporating at least a portion of the organic solvent fromthe emulsification mixture after the addition of the second portion ofthe aqueous phase. Moreover, in some aspects, the desired pH is a basicpH. For example, the basic pH comprises a pH with a range of pHs that isgreater than physiologic pH (a pH of around 7.4). In some embodiments,the basic pH is within a range of about 8 to about 14. In other aspects,the basic pH is around 10. In other embodiments, the pH is an acidic pHin a range of pHs that is less than physiologic pH. For example theacidic pH is within a range of about 1 to about 7. In other embodiments,the desired pH can be any pH that increases the solubility of the activeingredient within the aqueous phase.

In a non-limiting embodiment, the nanoparticles are prepared byemulsification, for example by forming a pre-emulsion organic phasecomprising the amphiphilic polymer and the organic solvent; optionally,adding the active ingredient to the pre-emulsion organic phase;combining the pre-emulsion organic phase with a pre-emulsion aqueousphase to form a pre-emulsion mixture; and emulsifying the pre-emulsionmixture to form an emulsion.

In certain embodiments, the organic solvent comprises a solvent that iswater miscible or water immiscible. For example, in those embodimentswhere the organic solvent is generally water immiscible, the organicsolvent comprise at least one of the following water-immisciblesolvents: dichloromethane (methylene chloride), chloroform, carbontetrachloride, dichloroethane, diethyl ether, ethyl acetate, andtoluene. In other embodiments where the solvent comprises awater-miscible solvent, the solvent, for example, comprises at least oneof the following solvents: acetaldehyde, acetic acid, acetone,acetonitrile, cyclohexane, ethanol dimethyl formamide, dioxane, heptane,hexane, methanol, formic acid, ethylamine, ethylene glycol, dimethylsulfoxide, glycerol, pentane, propanol, pyridine, tetrahydrofuran, andwater.

Emulsification can employ any conventional emulsification procedures toemulsify the aqueous and organic phases. For example, in someembodiments, the emulsification step can comprise methods such assonication and mechanical shearing (e.g., vigorous movement, such asstirring or homogenization with blades).

In some embodiments, prior to the addition to the organic phase, theactive ingredient can be at least partially dissolved in a carrier, suchas dimethyl sulfoxide. Moreover, in some embodiments, the activeingredient comprises an ionizable composition. For example, theionizable composition is a therapeutic, such as a histone deacetylaseinhibitor (e.g., quisinostat). In some aspects, the histone deacetylaseinhibitor is used to treat one or more forms of cancer. In otherembodiments, the histone deacetylase inhibitor is used to treat anyother disease associated with aberrant histone deacetylase activity.

In particular embodiments, the surfactant or stabilizer comprises atleast one of sodium cholate, sodium dodecyl sulphate, poloxamer, one ormore Tween® compounds (Croda International of East Yorkshire, UnitedKingdom), vitamin E tocopheryl polyethylene glycol succinate, andpolyvinyl alcohol. Moreover, the polymer may be an amphiphilic polymerselected from the group consisting of poly(lactic acid)-poly(ethyleneglycol), poly(lactic-co-glycolic acid)-poly(ethylene glycol),poly(lactic-co-glycolic acid)-d-α-tocopheryl polyethylene glycolsuccinate, poly(lactic-co-glycolic acid)-ethylene oxide fumarate,poly(glycolic acid)-poly(ethylene glycol),polycaprolactone-poly(ethylene glycol), or any salts thereof. Asprovided above, the polymer can also be a hydrophobic and/or hydrophilicpolymer.

The nanoparticles can also be prepared by nanoprecipitation. Thenanoparticles can also be prepared in the presence of the activeingredient.

The therapeutic nanoparticles herein preferably comprise a biologicallyactive ingredient and a nanoparticle, wherein the nanoparticle is anamphiphilic polymeric nanoparticle and the active ingredient comprisesan ionizable group and has a partition coefficient of log P>0.Non-limiting examples of suitable ionizable groups include: hydroxamicacid group, carboxyl group, hydroxyl group, sulfhydryl group, phenolicgroup, amino group, imidazole group, guanidinium group, sulphonamidegroup, and imide group. The therapeutic nanoparticle preferablycomprises at least 4%, more preferably at least 6%, or even morepreferably, at least 9% of the active ingredient (% w/w).

In certain non-limiting embodiments, the active ingredient is at leastpartially loaded onto the surface of the polymeric nanoparticle, e.g.,at least 30%, 60%, or 90%. The amphiphilic polymer in certainembodiments comprises PLA-PEG and has a weight averaged molecular weightof 2,000 to 60,000 daltons.

In certain embodiments the active ingredient comprises a histonedeacetylase inhibitor, e.g., vorinostat (SAHA), istodax, belinostat,apicidin, SBHA, scriptaid, sodium butyrate, trichostatin A, entinostat,panobinostat, mocetinostat, romidepsin, tubastatin A, givinostat,dacinostat, quisinostat, pracinostat, droxinostat, abexinostat,ricolinostat, tacedinaline, tubacin, resminostat, citarinostat,santacruzamate, nexturastat A, tasquinimod, parthenolide, and anypharmaceutically acceptable salts thereof.

The hydrodynamic diameter of the therapeutic nanoparticle is preferablybetween 20-300 nm, e.g., 50-200 nm. Furthermore, preferably thetherapeutic nanoparticle has a zeta potential of between −35 and +10 mV,more preferably between −10 and +10 mV.

In a particular non-limiting embodiment, the therapeutic nanoparticlecomprises a second biologically active ingredient, wherein the secondactive ingredient is encapsulated in the polymeric nanoparticle, e.g.,entrapped in the polymeric nanoparticle.

The invention is also directed to the use of the therapeuticnanoparticles described herein, in the manufacture of a medicament forthe treatment of a disorder and also to a method of treating a subjecthaving a disorder, e.g., cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the novel pH driven approach for achieving highquisinostat loading of PLA-PEG NPs. Deprotonation of the hydroxamic acidgroup of quisinostat at pH 10 increases electrostatic interactionbetween quisinostat and the surface of the PLA-PEG NPs.

FIG. 2 depicts QNPs imaged by TEM. QNPs appeared spherical, and their˜100 nm size is consistent with DLS measurements. No evidence of drugprecipitates in the samples was observed. Scale bar=0.2 μm.

FIG. 3 depicts the correlation between Nanoparticle size and quisinostatloading. Nanoparticle's hydrodynamic diameter, as measured by DLS,positively correlated (Pearson coefficient=0.9108, p<0.0001) with thequisinostat loading for each batch. Each data point represents anindividual batch.

FIG. 4 depicts in vitro quisinostat release from QNPs. QNPs releasedquisinostat into PBS at 37° C. over 48 h, with nearly 50% releaseoccurring in the first 6 h. Free quisinostat was completely releasedfrom the cassette within 4 h. Points and error bars represent themean±SD of 3 samples read in triplicate at each time point.

FIG. 5 depicts in vitro QNP efficacy against GL261. QNP and freequisinostat exhibited equipotent growth inhibition against GL261 murineglioma cells in vitro with IC50s of 30 and 24 nM, respectively. Pointsand error bars represent the mean±SD of 3 samples read in triplicate ateach dilution.

FIG. 6. Mice receiving QNP treatment showed similar weight fluctuationsover the course of treatments as control mice. Control treated miceweight remained steady until the tumor burden became too great. Errorbars indicate mean±SD (n=3-4 mice/treatment).

FIG. 7 depicts free quisinostat treatment efficacy in mice bearingorthotopic GL261 tumors. (A) Tumor growth was determined by the changein tumor size (mean±SD) from day 6, as measured by bioluminescence. (B)Survival is shown on the Kaplan-Meier plot. (C) Saline (n=5) and FreeQuisinostat (n=5) treated tumors both doubled in size every 2.4 days andhad median survival times of 22 and 19 days, respectively.

FIG. 8. QNP in vivo treatment efficacy in mice bearing orthotopic GL261tumors. (A) Tumor growth determined by the change in tumor size(mean±SD) from day 6 as measured by bioluminescence. (B) Survival isshown on the Kaplan-Meier plot. (C) Saline (n=4) and BNP (n=3) treatedtumors grew exponentially and had median survival times of 21.5 and 21days, respectively. QNP (n=4) treatment significantly slowed tumordoubling compared to both controls, leading to the significantlyprolonged survival of 27.5 days compared to BNP treatment. #designatessignificance (p<0.05) compared to BNP. * designates significance(p<0.05) compared to saline. Statistical testing on tumor doubling timewas performed with a one-way ANOVA followed by Tukey post-hoc testing.Statistical testing on survival was performed by the Mantel-Cox test.

DETAILED DESCRIPTION

Additional objectives, advantages, and novel features will be set forthin the description which follows or will become apparent to thoseskilled in the art upon examination of the detailed description whichfollows.

As provided in greater detail herein, the disclosure provides drugdelivery compositions, methods of fabrication, use of drug deliverycompositions in the manufacture of a medicament, and methods ofadministration. In some embodiments, the disclosure comprises amethodology of the fabrication of a drug delivery composition. In otherembodiments, the disclosure comprises a methodology of theadministration of a drug delivery composition for the treatment of oneor more diseases or disorders.

The drug delivery composition or method disclosed herein at leastpartially rely on and incorporate one or more aspects of nanotechnology.In some embodiments, the drug delivery composition comprises atherapeutic nanoparticle. As used herein, the term “therapeuticnanoparticle” refers to therapeutics in nanoparticle systems having thepotential to increase drug-loading capabilities, improve site-specificdelivery, controlled release, or a combination thereof. Therapeutics innanoparticle systems have been shown to improve drugs pharmacokineticsthrough prolonged circulation, passive accumulation in the target site,and prolonged drug release.

Biologically Active Ingredient

In some aspects, the therapeutic nanoparticle comprises a biologicallyactive ingredient. As used herein, “a biologically active ingredient”includes a compound, a molecule, a composition, a structure, and anelement, etc. In some embodiments, the biologically active ingredientincludes a therapeutics capable of treating a disease or a disorder.Non-limiting examples of the disease or disorder include neoplasticdiseases such as cancer, neurodegenerative diseases, multiple sclerosis(MS), diabetes, HIV, tuberculosis, psoriasis, arthritis, asthma,ischemic related diseases, eye diseases, steroids deficiencies, andaddictions, etc.

Non-limiting examples of cancer include solid tumors and blood-bornecancers, etc. Non-limiting examples of solid tumor include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma, leiomyosarcoma,synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer,colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breastcancer, ovarian cancer, prostate cancer, esophageal cancer, stomachcancer, oral cancer, nasal cancer, throat cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, uterine cancer, testicular cancer, small cell lung carcinoma,bladder carcinoma, lung cancer, epithelial carcinoma, glioma,glioblastoma multiforme, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, skin cancer, melanoma,neuroblastoma, retinoblastoma, and lymphangioendotheliosarcoma, etc.Non-limiting examples of blood-borne cancer include acute lymphoblasticleukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblasticT-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocyticleukemia (APL), acute monoblastic leukemia, acute erythroleukemicleukemia, acute megakaryoblastic leukemia, acute myelomonocyticleukemia, acute nonlymphocyctic leukemia, acute undifferentiatedleukemia, chronic myelocytic leukemia (CML), chronic lymphocyticleukemia (CLL), hairy cell leukemia, multiple myeloma, lymphoblasticleukemia, myelogenous leukemia, lymphocytic leukemia, myelocyticleukemia, Hodgkin's disease, non-Hodgkin's Lymphoma, Waldenstrom'smacroglobulinemia, Heavy chain disease, and Polycythemia vera, etc.

Non-limiting examples of the neurodegenerative disease includeHuntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease(PD), motor neuron disease, Schizophrenia, and amyotrophic lateralsclerosis (ALS), etc. Non-limiting examples of ischemic related diseasesinclude glaucoma, retinopathy, and macular degeneration, etc.Non-limiting examples of addiction include alcohol addiction andnicotine addiction, etc.

In some embodiments, the biologically active ingredient includes, forexample, a histone deacetylase inhibitor (HDACi). As is known in theart, HDACs are overexpressed in many types of cancers. Overexpression ofHDACs induces histone deacetylation and in turn, chromatin compaction.Chromatin compaction can further result in transcriptional suppressionof key genes involved in the prevention or suppression of tumorigenesis.As such, inhibition of HDAC activities via the administration ofHDACi(s) can reduce inhibition of tumor-suppressing genes, leading toimproved tumor suppression. Furthermore, aberrant HDAC activity orexpression has also been shown to cause non-cancerous diseases.

Non-limiting examples of HDACi include quisinostat, vorinostat (SAHA),istodax, belinostat, apicidin, SBHA, scriptaid, sodium butyrate,trichostatin A, entinostat, panobinostat, mocetinostat, romidepsin,tubastatin A, givinostat, dacinostat, pracinostat, droxinostat,abexinostat, ricolinostat, tacedinaline, tubacin, resminostat,citarinostat, santacruzamate, nexturastat A, tasquinimod, parthenolide,any pharmaceutically acceptable salts of any of the foregoing, and anyderivatives of any of the foregoing, etc. In some aspects, thebiologically active ingredient includes quisinostat, a derivative orsalt thereof.

As used herein, the term “derivative” refers to a compound that issynthesized from a parent compound by replacement of one atom withanother atom or group of atoms. Non-limiting examples of derivativeinclude a salt, a pharmaceutically acceptable salt, and chemicalmodifications with a group including but not limited to esters,fluorine, methoxy, ethyl, butyl, propyl, hexyl, or other organicmoieties.

In certain non-limiting embodiments, a therapeutic nanoparticlecomprising an HDACi is used for the treatment of cancer, e.g.,hematological cancer or a solid tumor. In other embodiments, atherapeutic nanoparticle comprising an HDACi is used for the treatmentof a non-cancerous disease. In some non-limiting embodiments, atherapeutic nanoparticle comprising quisinostat is used for thetreatment of hematological cancer, a solid tumor, or both. In yetfurther non-limiting embodiments, a therapeutic nanoparticle comprisingquisinostat is used for the treatment of glioma, for example,glioblastoma multiforme (“GBM”).

In some aspects, the active ingredient is water insoluble. As usedherein, the term “water insoluble” refers to an active ingredient havinga partition coefficient (log P) of at least 0. In some embodiments, theactive ingredient, for example, HDACi, has a log P selected from thegroup consisting of at least 0, at least 0.3, at least 0.7, at least 1,at least 1.3, at least 1.7, at least 2, at least 2.5, and at least 3. Inother embodiments, log P is between 0 and 3, or any number range inbetween, e.g., 0-2.6, 0.1-2.6, 0.1-2.2, 0.2-2.2, 0.2-1.8, 0.4-1.8 or0.4-1.4. In yet other embodiments, the active ingredient, for example,HDACi, has a log P selected from the group consisting of between 0 and2, or any number range in between, e.g., 0-1.8, 0.2-1.8, 0.2-1.6,0.4-1.4, 0.4-1.2, 0.4-1, or 0.5-2.

In some aspects, the water solubility of the active ingredient is, e.g.,less than 1 mg/ml, less than 0.5 mg/ml, less than 0.2 mg/ml, less than0.15 mg/ml or less than 0.1 mg/ml. In some embodiments, the watersolubility of the active ingredient is between 0.01 and 1 mg/ml, or anynumber range in between, e.g., 0.02-1 mg/ml, 0.02-0.8 mg/ml, 0.04-0.8mg/ml, 0.04-0.6 mg/ml, 0.05-0.6 mg/ml, 0.05-1 mg/ml or 0.05-0.5 mg/ml.As used herein, the term “water solubility” refers to the solubilitiesof the active ingredient in water, at a pressure of 1 atm and at roomtemperature (approx. 293.15 K).

In some embodiments, the active ingredient is an ionizable compoundincluding an ionizable group. As used herein, “ionizable” refers tocapable of dissociating atoms or molecules into electrically chargedspecies; “an ionizable compound” refers to any molecule, composition,structure, element, etc., that, under certain conditions, having one ormore atoms or molecules dissociated therefrom and form electricallycharged compounds, radicals, or both; and “ionizable group” refers to anuncharged group act as proton-donor or proton acceptor influencing thecapacity for a molecule to act as an acid or base. Non-limiting examplesof the ionizable group include a hydroxamic acid group, a hydroxylgroup, a carboxyl group, a sulfhydryl group, a phenolic group, an aminogroup, an imidazole group, a guanidinium group, a sulphonamide group,and an imide group, or a combination thereof. In some aspects, theactive ingredient (e.g., an HDACi) has a carboxyl group, a hydroxamicacid group, or both. For example, Quisinostat comprises a hydroxamicacid group.

In some embodiments, the active ingredient is 100% ionized in theaqueous phase. In other embodiments, the active ingredient is partiallyionized. As used herein, “partially ionized” refers to less than 100%ionized in the aqueous phase. For example, the active ingredient isbetween 10% and 99% ionized, or any percent range in between, e.g.,10-90%, 20-90%, 20-80%, 40-80% or 50-90%. In some aspects, the activeingredient is at least 20%, at least 50%, at least 70% or at least 90%ionized in the aqueous phase.

In other embodiments, the ionization of the active ingredient isincreased in the aqueous solution compared to neutral pH, by between 10%to 90%, or any percent range in between, e.g., increased by about 20%(e.g., 10-30%), by about 30% (e.g., 20-40%), by about 40% (e.g.,30-50%), by about 50% (e.g., 40-60%), by about 60% (e.g., 50-70%) or byabout 70% (e.g., 60-80%).

Under some circumstances, the ionization state of a specific ionizablegroup of the biologically active ingredient is critical for watersolubility of the active ingredient. In some embodiments, the specificionizable group is between 50% and 100% ionized in the aqueous phase, orany percent range in between, e.g., 50-90%, 60-90%, 60-80% or 70-80%. Inother embodiments, the specific ionizable group is at least 50%, atleast 60% or at least 70% ionized in the aqueous phase. In yet otherembodiments, the ionization of the active ingredient is increased in theaqueous solution compared to neutral pH, by between 10% to 90%, or anypercent range in between, e.g., increased by about 20% (e.g., 10-30%),by about 30% (e.g., 20-40%), by about 40% (e.g., 30-50%), by about 50%(e.g., 40-60%), by about 60% (e.g., 50-70%) or by about 70% (e.g.,60-80%).

In further non-limiting embodiments, the hydroxamic acid group ofquisinostat is between 50% and 100% ionized in the aqueous phase, or anypercent range in between, e.g., 50-90%, 60-90%, 60-80% or 70-80%. Inother embodiments, the hydroxamic acid group of quisinostat is at least50%, at least 60% or at least 70% ionized in the aqueous phase. Infurther embodiments, the ionization of the hydroxamic acid group isincreased in the aqueous solution compared to neutral pH, by between 10%to 90%, or any percent range in between, e.g., increased by about 15%(e.g., 5-25%), by about 25% (e.g., 15-35%), by about 35% (e.g., 25-45%),by about 50% (e.g., 40-60%), by about 60% (e.g., 50-70%) or by about 70%(e.g., 60-80%).

Some embodiments of the disclosure comprise adding the active ingredientto a solvent. Selection of the solvent is, at least in part, based onthe chemical structure of the active ingredient. As used herein, theterm “solvent” refers to any suitable liquid, compound, or molecule thatfunctions to solubilize the active ingredient in a state. In someaspects, the active ingredient is partially solubilized in the solvent.In other aspects, the active ingredient is completely solubilized in thesolvent.

Non-limiting examples of the solvent include dimethyl sulfoxide (DMSO),acetonitrile, acetone, and a combination thereof. In some embodiments, ahydrophobic active ingredient is added to DMSO, acetonitrile or acetone.In other embodiments, an HDACi is added to DMSO or acetone. In yet otherembodiments, quisinostat is added to DMSO.

In some embodiments, the therapeutic nanoparticle comprises a singleactive ingredient. In other aspects, the single active ingredient isconfigured as a hybrid molecule, such that one active ingredientpossesses different functionalities. In yet other aspects, thetherapeutic nanoparticle comprises two or more active ingredients. Insome embodiments, the second active ingredient is encapsulated in thetherapeutic nanoparticle, for example, the polymeric nanoparticle. Inother embodiments, the second active ingredient is entrapped in thepolymeric nanoparticle. In yet other embodiments, the second activeingredient is dissolved in the polymeric nanoparticle. In furtherembodiments, the second active ingredient is associated with ornon-covalently interacting with the polymeric nanoparticle. In yetfurther embodiments, the second active ingredient is loaded into, loadedonto or precipitated onto the polymeric nanoparticle.

Nanoparticle (NP) Preparation

As described herein, at least a portion of the drug delivery compositioncomprises one or more nanoparticles. Techniques used to preparenanoparticles include but are not limited to the spontaneous formationof nanoparticles (e.g., salting out or nanoprecipitation), emulsiondiffusion, emulsion evaporation, precipitation polymerization, emulsionand microemulsion polymerization, and interfacial polymerization.

Nanoparticles for drug delivery include numerous architectural designsin terms of size, shape, and materials. These include dendrimers,micelles, nanospheres, nanocapsules, fullerenes and nanotubes, andliposomes, etc. It is known in the art that the characteristics of eachparticle differ in terms of drug loading capacity, particle and drugstability, drug release rates, and targeted delivery ability. In certainembodiments, the nanoparticles are fabricated using conventionalcomponents, such as a solid particle (e.g., an Au- or Fe-based corenanoparticle), a liposome (or other lipid-derived materials), a micelle,a reverse micelle, or a microsphere, etc.

In some aspects, the nanoparticles are fabricated using one or morepolymer-based nanoparticles (polymeric nanoparticles), selected inaccordance with the anticipated use and the type and structure of theactive ingredient used therewith.

In some embodiments, the polymeric nanoparticles comprise a hydrophobicpolymer. In other embodiments, the polymeric nanoparticles comprise ahydrophilic polymer. In yet other embodiments, the polymericnanoparticles comprise an amphiphilic polymer. In some aspects, thepolymeric nanoparticles comprise substantially all hydrophobic polymers.In other aspects, the polymeric nanoparticles comprise substantially allhydrophilic polymers. In yet other embodiments, the polymericnanoparticles comprise a combination, e.g., a hydrophilic polymer and anamphiphilic polymer.

Non-limiting examples of the types of the amphiphilic polymer includeamphiphilic copolymer (e.g., a copolymer of a hydrophilic block coupledwith a hydrophobic block), amphiphilic graft copolymer, amphiphilicblock copolymer and amphiphilic random copolymer, etc.

Non-limiting examples of the amphiphilic polymer include poly(lacticacid)-poly(ethylene glycol) (PLA-PEG), poly(lactic-co-glycolicacid)-poly(ethylene glycol) (PLGA-PEG), poly(lactic-co-glycolicacid)-d-α-tocopheryl polyethylene glycol succinate,poly(lactic-co-glycolic acid)-ethylene oxide fumarate, poly(glycolicacid)-poly(ethylene glycol), polycaprolactone-poly(ethylene glycol), anysalts of the foregoing, and any derivatives of the foregoing, etc.

In some non-limiting embodiments, the amphiphilic polymer comprisesPLA-PEG, PLGA-PEG or any derivatives or salts thereof. In someembodiments, the HDACi-loaded nanoparticles comprise PLA-PEG, PLGA-PEG,or both. In other embodiments, the quisinostat-loaded nanoparticlescomprise PLA-PEG or any derivatives or salts thereof.

In some aspects, the PLA-PEG has a weight averaged molecular weight ofbetween 2,000 and 60,000 daltons, or any number range in between, e.g.,3,000-60,000, 3,000-50,000, 5,000-50,000, 5,000-40,000, 8,000-40,000,8,000-30,000, or 10,000-20,000 daltons.

In some aspects, the PLA-PEG block co-polymer comprises polymer chainhaving an about 16 k Da (e.g., 15 k to 17 k Da) PLA segment attached toan about 5 k Da (e.g., 4 k to 6 k Da) PEG segment. In other aspects, thePLA-PEG block co-polymer comprises polymer chain having an about 20 k Da(e.g., 19 k to 21 k Da) PLA segment attached to an about 5 k Da (e.g., 4k to 6 k Da) PEG segment.

In other embodiments, the amphiphilic polymer includes PLA and PEG.

As used herein, “PLA” refers to a polymer derived from the condensationof lactic acid or by the ring opening polymerization of lactide. In someaspects, the weight averaged molecular weight of PLA is between 5,000and 35,000 daltons, or any number range in between, e.g., 5,000-30,000,8,000-30,000, 8,000-25,000, 11,000-25,000, 11,000-21,000, 14,000-21,000,14,000-19,000, 15,000-17,000, or 17,000-19,000 daltons. In otheraspects, the PLA has a weight averaged molecular weight of about 16,000daltons (e.g., 15,000-17,000 daltons) or about 20,000 daltons (e.g.,between 19,000-21,000 daltons).

In some embodiments, the weight averaged molecular weight of PEG isbetween 1,000 and 10,000 daltons, or any number range in between, e.g.,1,000-9,000 daltons, 2,000-9,000 daltons, 2,000-8,000 daltons,3,000-8,000 daltons, 3,000-7,000 daltons, 4,000-7,000 daltons, or4,000-6,000 daltons. In other embodiments, the weight averaged molecularweight of PEG is about 5,000 daltons, for example, between 4,500 and5,500 daltons or between 4,000 and 6,000 daltons.

In some aspects, the ratio of PLA and PEG (PLA:PEG) is between 50:5 and10:5, or any number range in between, e.g., about 40:5, about 35:5,about 30:5, about 20:5 or about 16:5.

In yet other embodiments, the polymeric nanoparticles comprise two ormore amphiphilic polymers.

Organic Phase

The method of fabricating the therapeutic nanoparticles comprises mixingan organic phase with an aqueous phase. In some aspects, the organicphase is formed by combining a polymer with an organic solvent. In otheraspects, the organic phase is formed by combining a nanoparticle, forexample, a solid/non polymer nanoparticle (e.g., iron oxide core), withan organic solvent.

In some embodiments, the polymer is mixed in the organic solvent. Inother embodiments, the polymer is dissolved in the organic solvent. Inparticular non-limiting embodiments, an amphiphilic polymer, such aspoly(lactic acid)-poly(ethylene glycol) (i.e., PLA-PEG and/or PLA-b-PEG)is dissolved in an organic solvent, such as DCM. In further non-limitingaspects, the organic phase comprising the amphiphilic polymer is anemulsion.

In some non-limiting embodiments, the nanoparticles are prepared usingemulsification, and the organic solvent includes a water-immisciblesolvent. Non-limiting examples of the water-immiscible solvent includedichloromethane (DCM, methylene chloride), chloroform, carbontetrachloride, dichloroethane, diethyl ether, ethyl acetate, andtoluene, etc. In some aspects, for example, the organic phase comprisesPLA-PEG nanoparticles and DCM. In other non-limiting embodiments, thenanoparticles are prepared using emulsification, and the organic solventincludes water-miscible mixed with water-immiscible solvents. In yetother non-limiting embodiments, the nanoparticles are prepared usingnanoprecipitation, and the organic solvent includes a water-misciblesolvent. Non-limiting examples of the water-miscible solvent includeacetaldehyde, acetic acid, acetone, acetonitrile, cyclohexane, ethanol,dimethylformamide, dioxane, heptane, hexane, methanol, formic acid,ethylamine, dimethyl sulfoxide, pentane, propanol, pyridine, andtetrahydrofuran, etc.

In some aspects, a physical force (e.g., mixing, vortexing, or shaking)is applied to the polymer-organic solvent mixture to dissolve thepolymer in the organic solvent. In other aspects, the amphiphilicpolymer will go into solution without the addition of any significant ormaterial physical force.

Aqueous Phase

In certain non-limiting embodiments, the aqueous phase comprises one ormore hydrophilic solvents (e.g., water).

In some embodiments, the aqueous phase comprises a surfactant. As usedherein, the term “surfactant” refers to any substance that tends toreduce the surface tension between two different molecules. For example,between two liquids or between a liquid and a solid (e.g., the aqueousphase and the active ingredient). Non-limiting examples of thesurfactant include sodium cholate, sodium dodecyl sulphate, poloxamer,one or more Tween® compounds, vitamin E tocopheryl polyethylene glycolsuccinate, and polyvinyl alcohol, etc. Some aspects of the disclosureinclude dissolving the surfactant in the aqueous phase using a physicalforce (e.g., mixing, vortexing, or shaking). Other aspects of thedisclosure require no significant or material physical force fordissolving the surfactant in the aqueous phase. In some aspects, thesurfactant acts as an emulsifier to provide for a mixing of the organicphase and the aqueous phase.

In other embodiments, the aqueous phase comprises a stabilizer. As usedherein, the term “stabilizer” refers to any substance capable ofinhibiting the separation of the organic phase and the aqueous phase.Non-limiting examples of the stabilizer include sodium cholate, sodiumdodecyl sulphate, poloxamer, one or more Tween® compounds, vitamin Etocopheryl polyethylene glycol succinate, and polyvinyl alcohol, etc.Some aspects of the disclosure include dissolving the stabilizer in theaqueous phase using a physical force (e.g., mixing, vortexing, orshaking). Other aspects of the disclosure require no significant ormaterial physical force for dissolving the stabilizer in the aqueousphase. In some aspects, the stabilizer acts as an emulsifier to providefor a mixing of the organic phase and the aqueous phase.

In yet other embodiments, the aqueous phase comprises a surfactant and astabilizer.

In certain non-limiting embodiments, the aqueous phase comprises sodiumcholate, TPGS or PVA. In other non-limiting embodiments, the aqueousphase comprises sodium cholate.

“Critical micelle concentration” (CMC) depends on temperature andsometimes pH, among other parameters. For sodium cholate, the “criticalmicelle concentration” values are roughly 8, 8, 9, 9, and 11 mmol dm⁻³at 293.2, 298.2, 303.2, 308.2, and 313.2 K. In certain non-limitingembodiments, the weight percent (% w/w) of sodium cholate is between0-1%, or any number range in between, e.g., 0.01-1%, 0.01-0.9%,0.02-0.9%, 0.02-0.8%, 0.05-0.8%, 0.05-0.6% or 0.1-0.6%.

Some embodiments of the disclosure include adjusting the pH of theaqueous phase to increase the water solubility of the active ingredient.In some aspects, pH of the aqueous phase is reduced to improve the watersolubility of the active ingredient, for example, by adding an acidicsolution such as hydrochloric acid. In other aspects, pH of the aqueousphase is increased to improve the water solubility of the activeingredient, for example, by adding a basic solution such as sodiumhydroxide. In further aspects, a buffer with appropriate pKa is added tocontrol the pH of the aqueous solution.

Drug Loading

In some embodiments, the active ingredient is added to the mixture ofthe organic phase and the aqueous phase after NP formation. In otherembodiments, the active ingredient is added to the mixture during theremoval step (i.e., when the organic solvent is partially removed). Inyet other embodiments, the active ingredient is added to the organicsolvent comprising the polymer.

As used herein, “encapsulation efficiency” of the active ingredient iscalculated using the following equation:

${{encapsulation}\mspace{14mu} {{efficiency}(\%)}} = {\frac{{mass}\mspace{14mu} {of}\mspace{14mu} {active}\mspace{14mu} {ingredient}\mspace{14mu} {in}\mspace{14mu} {NPs}}{{mass}\mspace{14mu} {of}\mspace{14mu} {active}\mspace{14mu} {ingredient}{\mspace{11mu} \;}{used}\mspace{14mu} {in}\mspace{14mu} {the}{\mspace{11mu} \;}{formulation}} \times 100}$

In some embodiments, the encapsulation efficiency of the activeingredient is between 50-100%, or any percent range in between, e.g.,55-100%, 55-90%, 60-95%, 60-90%, 65-95% or 65-90%. In other embodiments,the encapsulation efficiency of the active ingredient is at least 50%,at least 55%, at least 60%, at least 70% or at least 80%. In othernon-limiting embodiments, the encapsulation efficiency of HDACi isbetween 50-95%, between 55-85% or 60-80%. In other non-limitingembodiments, the encapsulation efficiency of quisinostat is between50-100%, between 55-90%, between 60-80% or at least 60%.

The Therapeutic Nanoparticles

As used herein, “content of the active ingredient” in the therapeuticnanoparticles (%, w/w) is calculated using the following equation:

${{active}\mspace{14mu} {ingredient}\mspace{14mu} {{content}\left( {\% \ {w/w}} \right)}} = {\frac{{mass}\mspace{14mu} {of}\mspace{14mu} {active}\mspace{14mu} {ingredient}\mspace{14mu} {in}\mspace{14mu} {NPs}}{{mass}\mspace{14mu} {of}\mspace{14mu} {NPs}\mspace{14mu} {recovered}} \times 100}$

In some embodiments, the content of the active ingredient in thetherapeutic nanoparticles is, for example, at least 2%, at least 5%, atleast 10%, at least 15%, or at least 20%. In other embodiments, thecontent of the active ingredient in the therapeutic nanoparticles isbetween 2.5-20%, or any percent range in between, e.g., 2.5-17%,2.5-14%, 2.5-11%, 4-19%, 4-16%, 4-13%, 6-18%, 6-15%, 6-12%, 8-17%, 8-14%or 8-11%.

In other non-limiting embodiments, content of HDACi in the polymericnanoparticles is between 5-15%, or any percent range in between, e.g.,5-13%, 5-11%, 6-14%, 6-12%, 7-15%, 7-12% or 8-12%.

In yet other embodiments, the content of quisinostat in the therapeuticnanoparticles is, for example, about 3% (e.g., 2-4%), about 5% (e.g.,between 3% and 7.5%), about 10% (e.g., between 7% and 13%) or about 15%(e.g., between 10% and 20%). In this context, “about” refers to ±30%.

In some non-limiting aspects, the content of quisinostat in thepolymeric nanoparticles is between 2-20%, or any percent range inbetween, e.g., 3-18%, 3-16%, 4-15%, 5-15%, 6-14%, 6-13%, 7-12% or 7-11%.

In some embodiments, the hydrodynamic diameter of the therapeuticnanoparticle is 20 to 300 nm, or any number range in between, e.g., 20to 250 nm, 40 to 250 nm, 40 to 200 nm, 80 to 200 nm or 80 to 150 nm.

In other embodiments, the hydrodynamic diameter of HDACi-loadedtherapeutic nanoparticle is about 50 nm (e.g., 20-80 nm), about 75 nm(e.g., 45-105 nm), about 100 nm (e.g., 70-130 nm), about 150 nm (e.g.,120-180 nm), about 200 nm (e.g., 170-230 nm) or about 250 nm (e.g.,220-280 nm).

In yet other non-limiting embodiments, the hydrodynamic diameter ofquisinostat-loaded PLA-PEG therapeutic nanoparticle is between 50-200nm, or any number range in between, e.g., 50-180 nm, 70-180 nm, 70-150nm, 90-150 nm or 90-120 nm.

In some embodiments, the zeta potential of the therapeutic nanoparticlesis −50 to +50 mV, or any number range in between, e.g., between −50 to+30 mV, between −35 to +20 mV, between −35 and +10 mV, between −20 and+15 mV, between −20 and +10 mV or between −10 and +10 mV.

In other non-limiting embodiments, the zeta potential of theHDACi-loaded polymeric nanoparticles is about −35 mV (e.g., between −50and −20 mV), about −15 mV (e.g., between −25 and −5 mV), about −10 mV(e.g., between −20 and 0 mV) or about −0 mV (e.g., between −10 and +10mV).

In some non-limiting aspects, the zeta potential of thequisinostat-loaded therapeutic nanoparticles is between −25 mV and 0 mV,between −20 mV and 0 mV, between −16 mV and 0 mV, or between −14 mV and0 mV. In other non-limiting aspects, the zeta potential of thequisinostat-loaded polymeric nanoparticles is about −20 mV (e.g.,between −30 and −10 mV), about −15 mV (e.g., between −25 and −5 mV) orabout −10 mV (e.g., between −20 and 0 mV).

In some embodiments, the active ingredient is partially loaded onto thesurface of the polymeric nanoparticle. In non-limiting aspects, of thetotal active ingredient loaded, the percentage of the active ingredientloaded onto the surface of the polymeric nanoparticle is between 50 and100%, or any percent range in between, e.g., 50-90%, 60-90%, 60-80% or70-80%. In other embodiments, the active ingredient is loaded close tothe surface of the polymeric nanoparticle. In yet other embodiments, theactive ingredient is loaded substantially close to the surface of thepolymeric nanoparticle.

In some embodiments, an HDACi is loaded close to the surface of thepolymeric nanoparticle, e.g., PLA-PEG. In other embodiments, an HDACi isloaded substantially close to the surface of the polymeric nanoparticle,e.g., PLA-PEG. In yet other aspects, an HDACi is loaded within thehydrated PEG layer. In further aspects, an HDACi is loaded within thePLA polymer phase.

Emulsification

By way of example only, some embodiments provided improved methods ofmanufacturing a drug delivery composition using an improvedemulsion-evaporation method of nanoparticle manufacture.

Organic Solvent

Some aspects comprise forming an organic phase comprising an amphiphilicpolymer and an organic solvent. In some embodiments, the organic solventincludes a water-immiscible organic solvent selected from the groupconsisting of DCM, chloroform, carbon tetrachloride, dichloroethane,diethyl ether, ethyl acetate, and toluene, etc. In other embodiments,the organic solvent includes water-miscible solvent mixed withwater-immiscible solvents. In yet other embodiments, the water-misciblesolvent is selected from the group consisting of acetaldehyde, aceticacid, acetone, acetonitrile, cyclohexane, ethanol, dimethylformamide,dioxane, heptane, hexane, methanol, formic acid, ethylamine, dimethylsulfoxide, pentane, propanol, pyridine, and tetrahydrofuran. In certainnon-limiting aspects, the organic phase is formed by combining DCM andPLA-PEG.

Pre-Emulsion Aqueous Phase

Some embodiments of the disclosure comprise combining the organic phasewith a pre-emulsion aqueous phase to form a pre-emulsion mixture. Incertain non-limiting embodiments, the pre-emulsion aqueous phasecomprises one or more hydrophilic solvents (e.g., water).

In some embodiments, the pre-emulsion aqueous phase comprises asurfactant selected from the group consisting of sodium cholate, sodiumdodecyl sulphate, poloxamer, one or more Tween® compounds, vitamin Etocopheryl polyethylene glycol succinate, and polyvinyl alcohol. Someaspects of the disclosure include dissolving the surfactant in thepre-emulsion aqueous phase using a physical force (e.g., mixing,vortexing, or shaking). Other aspects of the disclosure require nosignificant or material physical force for dissolving the surfactant inthe pre-emulsion aqueous phase. In some aspects, the surfactant acts asan emulsifier to provide for a mixing of the organic phase and thepre-emulsion aqueous phase.

In other embodiments, the pre-emulsion aqueous phase comprises astabilizer selected from the group consisting of sodium cholate, sodiumdodecyl sulphate, poloxamer, one or more Tween® compounds, vitamin Etocopheryl polyethylene glycol succinate, and polyvinyl alcohol. Someaspects of the disclosure include dissolving the stabilizer in thepre-emulsion aqueous phase using a physical force (e.g., mixing,vortexing, or shaking). Other aspects of the disclosure require nosignificant or material physical force for dissolving the stabilizer inthe pre-emulsion aqueous phase. In some aspects, the stabilizer acts asan emulsifier to provide for a mixing of the organic phase and thepre-emulsion aqueous phase. In yet other embodiments, the pre-emulsionaqueous phase comprises a surfactant and a stabilizer.

In certain non-limiting embodiments, the pre-emulsion aqueous phasecomprises sodium cholate, TPGS or PVA. In other non-limitingembodiments, the pre-emulsion aqueous phase comprises sodium cholate.

In some embodiments, the pre-emulsion aqueous phase and the aqueousphase are prepared separately. In other embodiments, the pre-emulsionaqueous phase and the aqueous phase originate from the same aqueoussolution. In further aspects, the pre-emulsion aqueous phase is mixedwith the organic phase prior to emulsification, and the aqueous phase ismixed with the resulting emulsification mixture.

In certain aspects, the pre-emulsion aqueous phase and the aqueous phaseare differentially modified. For example, in some embodiments, a higherconcentration of surfactant is added to the pre-emulsion aqueous phasethan to the aqueous phase.

Mixing the Organic Phase and the Pre-Emulsification Aqueous Phase

Some embodiments comprise mixing the organic phase and the pre-emulsionaqueous solution. For example, the pre-emulsion aqueous phase is placedin a receptacle, and a physical force (e.g., vortexing) is applied tothe pre-emulsion aqueous solution. In some embodiments, while vortexing,the organic phase is added dropwise to the pre-emulsion aqueous phaseuntil the two phases are in the same container to form thepre-emulsification mixture.

Mixing the Emulsion and the Aqueous Phase

In some aspects, the emulsion and the aqueous phase are mixed bystirring (e.g., using a stir bar on a magnetic plate). In yet otheraspects, the mixture between the emulsion and the aqueous phase isstirred in an environment (e.g., a fume hood) that enables evaporationof some or all of the organic solvent. In further aspects, a vacuum isapplied to facilitate evaporation of the organic solvent. In yet furtheraspects, thermal energy is applied to facilitate the evaporation of theorganic solvent.

Adding the Active Ingredient

In certain non-limiting embodiments, the active ingredient, for example,HDACi is added to the organic phase before nanoparticles are formedthrough emulsification. In other embodiments, the active ingredient(e.g., HDACi) is added to the emulsion (the organic phase afternanoparticles are formed through emulsification). In yet otherembodiments, for example, the active ingredient is added before removalof the organic phase (e.g., the evaporation step). In furtherembodiments, for example, the active ingredient is added during theevaporation step.

In some aspects, after the formation of the organic phase andpreparation of the active ingredient, these two elements are combined.In some aspects, the active ingredient (e.g., HDACi) is added in agenerally drop-wise manner into the organic phase. In further aspects, aphysical force (e.g., vortexing) is applied to the resulting mixture tocombine the active ingredient and the organic phase.

Removal of Organic Solvent

In some aspects, after removal of the organic solvent (e.g., byevaporation) and formation, the drug delivery composition is collectedand washed. For example, after evaporation, the resulting mixture isfiltered through a filter of desirable size (e.g., 0.22 μM) and theresulting filtrate is filtered again using filter tubes (e.g., 100kiloDalton cut-off) and centrifugation methodologies. In some aspects,after one or more filtrations and washes, the resulting drug deliverycomposition is mixed with a compound (e.g., trehalose) and stored in thedesired state (such as frozen or lyophilized) for storage and stability.

Emulsification

Some embodiments comprise emulsifying the pre-emulsification mixture. Insome aspects, emulsification comprises a chemical, a thermal, or amechanical action. For example, the mechanical action compriseshomogenization using a blade, sonication using an ultrasonicator probethat is at least partially submerged in the pre-emulsification mixture,multiple (e.g., two or more) bursts of ultrasonication lasting severalseconds, or a combination thereof. By way of example only, the burstsmay last three or ten-seconds. Depending on the active ingredient,different numbers of bursts or different durations of bursts is used.

Certain non-limiting embodiments of fabricating a therapeuticnanoparticle comprising the steps of (a) preparing an aqueous phase; (b)adjusting the pH of the aqueous phase; (c) mixing an organic phasecontaining a polymeric nanoparticle with the aqueous phase; (d) adding awater-insoluble biologically active ingredient to the mixture; and (e)removing the organic solvent from the mixture; wherein the activeingredient having a higher water solubility in the adjusted pH than inneutral pH. In some aspects, adjusting the pH of the aqueous phaseincreases the electrostatic interaction between the active ingredientand the polymeric nanoparticles. In other aspects, at least 50%, atleast 65%, at least 70%, at least or at least 90% of the activeingredient is ionized in the aqueous phase. In yet other aspects, thepercentage of ionized active ingredient in the aqueous phase is between50% and 100%, or any percentage in between, e.g., 50-90%, 60-90%, 60-80%or 70-80%.

Other non-limiting embodiments of fabricating a therapeutic nanoparticlecomprising the steps of (a) forming an organic phase comprising anamphiphilic polymer and an organic solvent; (b) adding a biologicallyactive ingredient having an ionizable group; (c) combining the organicphase with a pre-emulsion aqueous phase to form a pre-emulsion mixture;(d) emulsifying the pre-emulsion mixture to form an emulsion; (e)combining the emulsion with an aqueous phase; and (0 evaporating theorganic solvent from the combination of the emulsion and the aqueousphase. In some embodiments, 50-100% of the active ingredient is ionizedin the aqueous phase. In other embodiments, the active ingredient isadded when the organic solvent is partially evaporated.

In some aspects, the active ingredient comprises an HDACi, for example,quisinostat. In other aspects, the pH of the aqueous phase is adjustedto increase the water solubility of the active ingredient. In certainnon-limiting aspects, the pH of the aqueous phase is adjusted to aboutpH 10 (e.g., pH 9-12) to increase the water solubility of quisinostat.

In some aspects, manufacturing a HDACi-loaded therapeutic nanoparticlecomprises: (a) forming an organic phase comprising a polymer (e.g.,PLA-PEG) and an organic solvent (e.g., dichloromethane); (b) adding theHDACi to the organic phase; (c) forming an aqueous phase comprising ahydrophilic solvent; (d) mixing together the organic phase and apre-emulsion aqueous phase to form an emulsification mixture; (e)emulsifying the emulsification mixture; (f) adjusting the pH of anaqueous phase to a desired pH; and mixing together the emulsificationmixture with the aqueous phase. In further aspects, the method furthercomprises evaporating at least a portion of the organic solvent from theemulsification mixture after the addition of the aqueous phase. In someaspects, increased percent ionization of a key ionizable group increasesthe solubility of the HDACi. In other aspects, the ionizable group isselected from the group consisting of a hydroxamic acid group, acarboxyl group, a hydroxyl group, a sulfhydryl group, a phenolic group,an amino group, an imidazole group, a guanidinium group, a sulphonamidegroup, and an imide group.

As used herein, the “desired pH” refers to a pH that increases thesolubility of the active ingredient (e.g., HDACi) in the aqueous phase.For example, when a compound's solubility is significantly impacted bythe percent ionization of its hydroxamic acid group, the desired pH isselected from the group consisting of between pKa+0.37 and pH14 (about70% ionization), between pKa+1 and pH14 (about 90% ionization), andbetween pKa+2 and pH14 (about 99% ionization). On the other hand when acompound's solubility is significantly impacted by the percentionization of an acidic group, the desired pH is selected from the groupconsisting of between pH0 and pKa−0.37 (about 70% ionization), betweenpH0 and pKa−1 (about 90% ionization), and between pH0 and pKa+2 (about99% ionization).

In some embodiments, manufacturing a quisinostat-loaded therapeuticnanoparticle comprises: (a) forming an organic phase comprising apolymer (e.g., PLA-PEG) and an organic solvent (e.g., dichloromethane);(b) adding quisinostat to the organic phase; (c) forming an aqueousphase comprising a hydrophilic solvent; (d) mixing together the organicphase and a pre-emulsion aqueous phase to form an emulsificationmixture; (e) emulsifying the emulsification mixture; (0 adjusting the pHof an aqueous phase to about pH 10 (e.g. pH 8-14, pH 9-13, or pH 9-11);and mixing together the emulsification mixture with the aqueous phase.In further aspects, the method further comprises evaporating at least aportion of the organic solvent from the emulsification mixture after theaddition of the aqueous phase. In yet further aspects, quisinostat isdissolved in a solvent, such as DMSO. In some embodiments, the aqueoussolvent comprises a surfactant, for example, sodium cholate, astabilizer, or both.

Additional Agent or Moiety

In some embodiments, the therapeutic nanoparticles further comprise anagent or a moiety. The agent is configured, for example, as acomposition, a molecule, structure, or a chemical. In some aspects, theagent is used during the administration of the drug delivery system tothe subject in need thereof. In other aspects, the agent is used afteradministration of the drug delivery system to the subject in needthereof.

Non-limiting examples of the agent include, for example, an imagingagent, a targeting agent, an agent that modifies the action or activityof the active ingredient, and a combination thereof, etc.

Non-limiting examples of the targeting agent include, for example, folicacid (FA), transferrin, aptamer, epidermal growth factorreceptor-targeting molecule, a peptide (e.g., a RGD peptide), and anantibody (e.g., an antibody or a portion of an antibody that targets adesired antigen), etc. (Steichen, Caldorera-Moore et al. 2013, Bazak,Houri et al. 2015). In some aspects, the targeting agent directs thedrug delivery composition, e.g., HDACi-loaded polymeric nanoparticle toa particular cell- or tissue-type, such as a tumor.

Non-limiting examples of the imaging agent include contrast medium(which absorbs or alters external electromagnetism or ultrasound) andradiopharmaceutical (which emits radiation), etc. As used herein, theterm “contrast agent” refers to a substance used to increase thecontrast of structures or fluids within the body in medical imaging.Non-limiting examples of the contrast agent include, for example,radiocontrast media, MRI contrast agents, and ultrasound contrastagents, etc. Radiopharmaceuticals are a group of pharmaceutical drugswhich have radioactivity and can be used as diagnostic and therapeuticagents. Non-limiting examples of Radiopharmaceutical include, forexample, calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57,cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3,indium-111, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m,nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82,samarium-153, selenium-75, sodium-22, sodium-24, technetium-99m,thallium-201, xenon-133, and yttrium-90, etc. Additional, non-limitingexamples of the imaging agent include, for example, a dye, afluorophore, a radioactive-based agent, and any other imaging agents,such as quantum dots, etc. In some embodiments, the imaging agent isused to visualize the local environment.

Use of Therapeutic NPs for the Treatment of a Disorder in a Subject

In some embodiments, the active ingredient is provided in atherapeutically effective amount. In further embodiments, the drugdelivery composition is administered to a subject in need thereof.

As used herein, the “therapeutically effective amount” refers to anyamount of the active ingredient that treats the subject, for example, adose or a concentration that provides a therapeutically effective amountof the active ingredient (e.g., HDACi).

The addition of a therapeutically effective amount of the activeingredient encompasses any method of dosing. Dosing of the activeingredient may include single or multiple administrations of the drugdelivery composition that includes the active ingredient.

Examples include administration of the drug delivery composition (e.g.,once or multiple administrations) for a period of time until adiminution of the disease state is achieved, preventative treatmentsapplied prior to the instigation of symptoms, or any other dosingregimen known in the art or yet to be disclosed that one skilled in theart would recognize as a potentially effective regimen. A final dosingregimen including the regularity of and mode of administration dependson a number of non-limiting factors such as the subject, the severity ofthe affliction, the route of administration, the stage of diseasedevelopment, the presence of other conditions such as pregnancy,infancy, or the presence of an additional disease; or any other factornow known or yet to be disclosed.

Determination of a therapeutically effective amount of the activeingredient is within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein. Theeffective amount of the active ingredient and/or the drug deliverycomposition used to affect a particular purpose as well as its toxicity,excretion, and overall tolerance may be determined in vitro, or in vivo,by pharmaceutical and toxicological procedures either known now by thoseskilled in the art or by any similar method yet to be disclosed. Oneexample is the in vitro determination of the IC₅₀ (half maximalinhibitory concentration) of the active ingredient in cell lines ortarget molecules. Another example is the in vivo determination of theLD₅₀ (lethal dose causing death in 50% of the tested animals) of theactive ingredient. The exact techniques used in determining an effectiveamount will depend on factors such as the type and physical/chemicalproperties of the active ingredient, the property being tested, andwhether the test is to be performed in vitro or in vivo. Thedetermination of an effective amount of a particular active ingredientwill be well known to one of skill in the art who will use data obtainedfrom any tests in making that determination.

As disclosed above and herein, the drug delivery system can be used totreat a disease or condition. As used herein, treatment of a conditionor disease is the practice of any method, process, or procedure with theintent of halting, inhibiting, slowing or reversing the progression of adisease, disorder or condition, substantially ameliorating clinicalsymptoms of a disease disorder or condition, or substantially preventingthe appearance of clinical symptoms of a disease, disorder or condition,up to and including returning the diseased entity to its condition priorto the development of the disease. Generally, the effectiveness oftreatment can be determined by comparing treated groups with non-treatedgroups. For example, some embodiments of the drug delivery system can beused to treat one or more forms of cancer.

Cancer cells include any cells derived from a tumor, neoplasm, cancer,pre-cancer, cell line, malignancy, or any other source of cells thathave the potential to expand and grow to an unlimited degree. One ormore cancer cells in the context of an organism may also be calledcancer, tumor, neoplasm, growth, malignancy, or any other term used inthe art to describe cells in a cancerous state.

As used herein, the subject includes any human or non-human mammal,including for example: a primate, cow, horse, pig, sheep, goat, dog,cat, or rodent, including any organisms capable of developing cancer,including human patients that are suspected of having cancer, that havebeen diagnosed with cancer, or that have a family history of cancer.

In some embodiments, the drug delivery composition can be administeredto a subject in need thereof. For example, as provided above, the drugdelivery composition can be delivered to a subject with cancer with theintention the drug delivery composition be used to treat cancer. In someembodiments, the drug delivery composition can be formed as anydesirable form, including tablets, including suspension tablets,chewable tablets, effervescent tablets or caplets; pills; powders suchas a sterile packaged powder, a dispensable powder, and an effervescentpowder; capsules including both soft or hard gelatin capsules such asHPMC capsules; lozenges; a sachet; a sprinkle; a reconstitutable powderor shake; a troche; pellets such as sublingual or buccal pellets;granules; liquids for oral or parenteral administration (e.g.,intravenous); suspensions; emulsions; semisolids; or gels. For example,in some embodiments, the drug delivery composition can be administeredin a generally liquid formulation that is provided at a generallyphysiological pH (e.g., around 7.4).

Methodologies of manufacture provided in general accordance with someembodiments may include methods of manufacturing of a drug deliverycomposition. As described herein, the methods of manufacture may beconsidered to be significant and surprising improvements overconventional methodologies. For example, in some embodiments, themethods of manufacture provided herein may comprise a significantimprovement over some common methods of manufacturing drug deliverycompositions, such as polymer-based nanoparticles. In particular, themethods of manufacture provided herein may be used with and/or as areplacement for conventional methods of manufacture of polymer-basednanoparticles, such as, but not limited to the emulsion-evaporationmethod, the emulsion-diffusion method, the nanoprecipitation method, andthe salting out method. Y. Wang et al., Nanomaterials 2016; 6, 26, whichis hereby incorporated by reference in its entirety for all purposes.

As mentioned above, the modified methods of manufacture contained hereinprovide significant improvements over the existing methodologies. Inparticular, the adjustment of the pH of the aqueous phase to a generallybasic pH can provide an altered physical environment, which can lead toan increased amount of active ingredient being loaded onto the formingor formed nanoparticles. Without being bound by any particular theory,it is believed that the basic pH of the aqueous phase creates anenvironment in which the active ingredient (e.g., any ionizablecompound) is ionized prior to loading. For example, the ionized activeingredient may be generally stabilized and in equilibrium with thehydrophilic and/or hydrophobic areas of the amphiphilic polymers thatform the therapeutic nanoparticles. In other aspects, it is alsopossible that the ionized active ingredient is generally precipitatedfrom solution and is then bound to an exposed surface of the resultingnanoparticle.

Regardless of the theory behind the formation, as a result of theimproved methodologies contained herein, the ionized active ingredientcan be non-covalently bound to an exposed surface of the resultingnanoparticle. Compared to conventional systems in which the activeingredient must be loaded within the forming nanoparticle to be capturedinside the nanoparticle and later delivered, this methodology results insignificantly more loaded active ingredient. Specifically, themethodologies detailed herein can provide approximately 4-10 fold moreloaded active ingredient compared to the conventional process.

EXAMPLES

Recent advances have highlighted the role of epigenetic aberrations inthe development and progression of many cancer types, includingglioblastoma (GBM) [1-6]. Histone deacetylases (HDACs) are a class ofenzymes capable of producing epigenetic modification of cellularbehavior. HDACs are responsible for the deacetylation of lysine residueson histones to regulate chromatin structure, transcription factorbinding sites and gene expression, and their overexpression has beenobserved in dedifferentiated, aggressively proliferating tumors [7-11].Importantly, molecules that inhibit HDACs (HDAC inhibitors, HDACis) arecapable of producing apoptosis and cell cycle arrest, and they alsosensitize cells to conventional DNA damaging treatments [12-16].Currently, three first-generation HDACis are clinically approved forcutaneous T-cell lymphoma [17]. However, despite promising preclinicalefficacy of first generation HDACis both in vitro and in vivo, clinicaltrials of HDACis have failed to show treatment benefits in solid tumors.It has been proposed that inadequate delivery and short biologicalhalflife of most HDACis contribute to their underwhelming in vivoefficacy [18, 19]. Second generation HDACis, like quisinostat, weredesigned and shown to be significantly more selective and potent againstclass I HDACs with a longer duration of action compared to firstgeneration HDACis, but these agents still failed to show significantefficacy as a monotherapy against solid tumors, presumably due to poortumor delivery [18, 20]. In previous work, it has been shown thatpolymeric nanoparticles (NPs) can effectively encapsulate poorly watersoluble small molecules to improve their tolerability in vivo anddelivery to intracranial GL261 GBM tumors, which enables effectivetreatment of tumors after intravenous administration [21]. Importantly,Wang et al. showed the encapsulation of quisinostat withinPLGA-lecithin-PEG core-shell NPs potentiated the effects of radiation insubcutaneous PC3 tumors more effectively than free drug [22]. Thus, thegoal of this disclosure was to develop a formulation process that wouldeffectively encapsulate quisinostat in NPs composed of PLA-PEG and totest whether encapsulated quisinostat would be capable of treatingorthotopic GBM. Through the process of developing this drug deliverycomposition, the inventors identified a novel, pH-driven approach forachieving high quisinostat loading. In contrast to traditional methodsthat improve drug encapsulation by decreasing the aqueous solubility ofthe drug to drive it into the polymer core, this novel method achieveshigh loading by improving the solubility of quisinostat in the aqueousphase prior to solvent evaporation.

Materials Quisinostat (JNJ-26481585) was obtained from APExBio (Houston,Tex. USA). Poly(d,l-lactide)-b-methoxy poly(ethylene glycol) (PLA-PEG,Mw ˜16 k:5 k Da or PLA-PEG, Mw ˜20 k:5 k) was purchased from PolySciTech(West Lafayette, Ind. USA). Endotoxin free (<0.0050 EU/ml) water fromG-Biosciences (St. Louis, Mo. USA) was used throughout nanoparticlefabrication. Dimethyl sulfoxide (DMSO), dichloromethane (DCM), sodiumcholate, lx phosphate buffered saline (PBS), hydrochloric acid (HCl,0.1001 M) and sodium hydroxide (NaOH, 0.1001 M) were all purchased fromSigma-Aldrich (St. Louis, Mo. USA). Dulbecco's modified Eagle medium(DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA and geneticinselective antibiotic (G-418) were purchased from Gibco Invitrogen(Carlsbad, Calif., USA). Greiner T25 tissue culture flasks with filtercap and Costar 96-well assay plates were purchased from VWRInternational (Radnor, Pa., USA). Beetle luciferin (potassium salt) andCellTiter-Glo Luminescent Cell Viability Assay were purchased fromPromega (Madison, Wis., UAS).

Nanoparticle fabrication The following steps of nanoparticle fabricationare included as an illustration only and are not intended to be limitingto the overall scope of the instant subject matter.

Nanoparticles were produced by a modified single emulsion-solventevaporation as previously reported [21,23,24]. 50 mg PLA-PEG dissolvedin 2 ml DCM was added dropwise into 4 ml of 1% (w/v) sodium cholatewhile vortexing, then probe sonicated (Fisher Scientific Model 705 SonicDismembrator, Waltham, Mass. USA) on ice in 3, 10-s bursts at 40%amplitude. The resulting emulsion was added to an evaporation phaseconsisting of 20 ml of 0.3% (w/v) sodium cholate (the second aqueoussolution) and allowed to stir for 3 h to evaporate the DCM.

Drug loading, Collection and Washing of Nanoparticles Drug loadednanoparticles were produced by adding 5 mg quisinostat, dissolved in 300μl DMSO, dropwise into the organic phase or the evaporation phase, asspecified for each formulation in Table 1. For nanoparticles made underbasic or acidic conditions, the pH of the 0.3% sodium cholateevaporation phase was adjusted to the specified pH by adding dilute (0.1M) NaOH or HCl. After the 3 h, nanoparticles were washed andconcentrated through Amicon Ultra-15 Centrifugal Filters (100 kDacut-off) for 4, 20 min spins at 5000 RCF. Aliquots were frozen andlyophilized to deter-mine nanoparticle concentration and drug loading.The rest of the nanoparticles were frozen and stored at −80° C.

Nanoparticle Characterization

Drug Loading Drug loading was quantified by absorbance (300 nm) on aTecan plate reader. Lyophilized nanoparticles were dissolved at 5 mg/mlin DMSO. The nanoparticle samples were plated in triplicate (40 μlnanoparticles and 10 μl DMSO per well) in a clear, flat bottom 96-wellassay plate. A control curve was constructed in technical triplicate byadding 40 μl blank nanoparticles per well and spiking with 10 μl ofknown drug concentrations in DMSO. Quisinostat loading was calculated asmass quisinostat/mass polymer (w/w %).

TABLE 1 Quisinostat % Zeta added Loading Diameter Potential Formulation(mg) O/E pH (w/w) (nm) PDI (mV) BNP 0 — 7 — 96.3 ± 2.08 0.1 ± 0.01  −13± 2.0 QNP-1 5 O 7  1.3 ± 0.71  101 ± 2.52 0.1 ± 0.01 −4.9 ± 2.3 QNP-2 5O 2 0.47 ± 0.25  103 ± 2.08 0.1 ± 0.01 −6.2 ± 1.7 QNP-3 5 O 10  5.0 ±0.51  113 ± 10.0 0.1 ± 0.01  −8.2 ± 0.53 QNP-4 5 E 10  9.3 ± 0.29  128 ±8.50 0.1 ± 0.02 −6.0 ± 1.0 QNP-5 5 E 7  2.7 ± 0.15  112 ± 2.52 0.1 ±0.01 −5.2 ± 1.7 QNP-6 7.5 E 10  9.9 ± 0.21  129 ± 4.51 0.1 ± 0.02 −9.8 ±1.1 QNP-7 10 E 10  7.7 ± 0.35  121 ± 2.31 0.1 ± 0.02 −9.4 ± 2.1 QNP-8 5E 9  5.3 ± 0.12  115 ± 5.29 0.1 ± 0.01 −8.2 ± 1.4 QNP-9 7.5 E 11  8.9 ±0.60  126 ± 3.21 0.1 ± 0.01 −7.8 ± 1.6

Size and Zeta Potential Nanoparticle hydrodynamic diameter and zetapotential were measured using the NanoBrook 90Plus Zeta (BrookhavenInstruments, Holtsville, N.Y. USA). All measurements were done at ananoparticle concentration of 0.1 mg/ml in triple filtered (0.2 μm) 1 mMKCl. Reported values represent the mean±standard deviation from 3batches unless otherwise indicated.

Transmission electron microscopy Transmission electron microscopy (TEM)measurements were measured on the Phillips CM 12 operated at anaccelerated voltage of 120 kV using 400 mesh formvar-coated copper gridsFCF400-Cu-SB (Electron Microscopy Sciences, PA, USA). Copper-grids werefirst glow-discharged to increase hydrophilicity on the surface. Sampleswere then diluted with DI water (final concentration 4 mg/ml). Sampleswere prepared by pipetting 3 μl of diluted solution to theglow-discharged grids followed by ambient drying using Whatman FilterPaper (Sigma Aldrich, USA).

Controlled Release Quisinostat release from nanoparticles was determinedusing a protocol adapted from Wang et al. [22]. Nanoparticles werediluted to 20 mg/ml in PBS (pH 7) and 400 μl was transferred to a 3.5 kMWCO Slide-A-Lyzer Dialysis cassette (Thermo Fisher Scientific, Waltham,Mass. USA) in triplicate. Each cassette was immersed in 2 l PBS (pH 7,replaced at each time point) at 37° C. with gentle stirring (100 rpm).At each time point, 30 μl nanoparticles was removed from the cassetteand dissolved in 150 μl DMSO. 60 μl dissolved nanoparticles was added intriplicate to a clear, flat bottom, 96-well plate, and the amount ofdrug remaining was quantified by absorbance as described above. A freequisinostat control at the equivalent concentration was included tomeasure quisinostat movement across the membrane using the sameprotocol.

Cell Culture GL261-LucNeo cells were generated by retroviraltransduction of parent GL261 cells. The LucNeo construct (obtained fromAndrewKung laboratory, Dana-Farber Cancer Institute) is described inRubin et al. [25]. Cells were maintained under normal adherent cultureconditions supplemented with G-418 as a selection pressure. Cells weregrown in T25 flasks in DMEM containing glucose, L-glutamine and 10% FBSat 37° C. and 5% CO₂. 0.25% trypsin-EDTA was applied to collect cells,and a Cellometer mini (Nexcelom Bioscience, Lawrence, Mass. USA) wasused to count cells prior to all in vitro and in vivo experiments.

In vitro Nanoparticle Efficacy GL261 cells were seeded in 96-well flat,white walled, clear bottom plates at a density of 3 k cells/well in 100μl media and allowed to attach for 4 hours prior to adding treatments.Each plate was treated with 10 μl/well of 19 serial dilutions (1:2)ranging from 10 to 0 μM in PBS of either free drug or nanoparticles.After 72 hours, cell viability was assessed using CellTiter-Glo, and anIC₅₀ value was calculated using GraphPad Prism (San Diego, Calif. USA)by a nonlinear fit of the log (inhibitor) vs. response function.

Tumor induction Orthotopic GL261-LucNeo tumors were induced in C57BL/6albino mice (Harlan Laboratories, Indianass, Ind., USA) as previouslyreported [23, 21]. Briefly, mice were anesthetized with anintraperitoneal injection of ketamine/xylazine (100/10 mg/kg) andmounted in a stereotaxic frame (Kopf Instruments, Tujunga, Calif., USA)on top of an infrared heating pad to maintain animal temperature. Theanimal's head was shaved and sterilized with three alternating passeseach of betadine and ethanol. A 1 cm incision was made over midline, anda burr hole was drilled 2 mm lateral, 0.1 mm posterior of bregma. Ahamiltion syringe (29 gauge needle) containing 75 k GL261-LucNeo cellsin 2 μl DMEM was inserted into the hole to a depth of 2.8 mm and thecells were injected over 2 min. The needle was left in place for 1 minto reduce backflow before the wound was closed with staples. All animalsreceived a subcutaneous (SQ) injection of Buprenorphine SR prior tosurgery, and ibuprofen was provided in their water ad lib for 1 week forpain.

Tumor growth Bioluminescence was used to monitor and measure tumorgrowth as previously described [21, 23]. Imaging was done on the XenogenIVIS Spectrum in vivo imaging system every 3-4 days starting at day 6after tumor implantation. Mice received a SQ injection of luciferin (150mg/kg) and were imaged 25 min post injection under 2% isoflurane. TheLiving Image software was used to draw an ROI around the tumor signaland measure the size of each tumor (total flux, photons/sec).

Tumor treatment Quisinostat-loaded nanoparticles were tested in vivo inmice bearing orthotopic GL261 tumors. After the first imaging, mice wererandomly assigned to a treatment group. For the free drug study, thisincluded saline control (100 μl) or free quisinostat (10 mg/kg IP,solubilized in 20% hydroxy-propyl-β-cyclodextrin, pH 8.7). For thenanoparticle drug study, this included saline (100 μl), blanknanoparticles (BNP, 1000 mg/kg polymer), or quisinostat-loadednanoparticles (QNP, 50 mg/kg quisinostat). One mouse in the nanoparticlestudy was excluded for lack of a tumor signal at the initial imaging.Mice were treated by intravenous injection (lateraltail vein) on days11, 12, 18, and 19 post tumor induction. Treatment efficacy was measuredby tumor growth and median survival. Mice were monitored daily andeuthanized at the sign of symptoms (lack of grooming, abnormal gait,hunched posture, etc.) or greater than 15% weight loss.

Statistics All statistical tests were performed using GraphPad Prism 5software. Particle localization regions were compared using a 2-wayANOVA. Tumor growth for each treatment was compared by fitting theaverage growth with an exponential curve fit and comparing treatmentsusing a one-way ANOVA. Survival differences were compared using aKaplan-Meier curve and the Mantel-Cox test.

Results

Nanoparticle Loading and Characterization NPs produced from amphiphilicpolymers such as PLA-PEG possess a hydrophobic core, which is utilizedas a favorable environment for the encapsulation of water-insolublesmall molecules [26, 27]. Our initial attempts to encapsulatequisinostat in PLA-PEG NPs followed a standard single emulsion-solventevaporation technique under neutral conditions. Quisinostat loaded NPs(QNPs) formed effectively. However, a relatively poor loading of 1.3%(Table 1, QNP-1) was achieved, which is comparable with prior reports of2.3% (w/w) quisinostat encapsulation within PLGA-lecithin-PEG core-shellNPs [22]. Attempts were made to improve loading by varying a number oftraditional formulation parameters known to affect drug loading (solventmixtures (acetonitrile, dimethylformamide, acetone, DMSO, DCM, ethylacetate), nanoprecipitation, feed ratios, and temperature) [28, 29].However, none of these changes brought quisinostat loading above 2%.

In an emulsion based approach to NP formation, a hydrophobic drug istypically dissolved with the polymer in an organic solvent to aid in theencapsulation of the drug during NP formation, followed by evaporationof the solvent. The final loading of drug within the NP is thought to bedetermined by diffusion of drug out of the polymer core after NPformation, which is directly related to the solubility of the drug inthe aqueous phase. Thus, one approach for improving loading of drugwithin NPs formed by emulsion is to fabricate particles under conditionsthat reduce drug solubility in the water phase, which is believed todrive partitioning of drug into the particle core [28, 30, 31]. Becausequisinostat exhibits increased water solubility at a basic pH, it washypothesized that acidifying the evaporation phase to pH 2 wouldincrease quisinostat loading. However, it was observed that drug loadingunder acidic conditions significantly decreased compared to NPs producedunder neutral conditions to 0.47% (QNP-2). As a negative control, theeffect of raising the evaporation phase pH to 10 was also tested.Interestingly, a basic evaporation pH resulted in significantly higherloading compared to NP produced at pH 2 or 7, achieving a loading of5.0% (QNP-3).

The observation that loading improves when quisinostat's aqueoussolubility is increased suggests a loading mechanism that does not relysolely on hydrophobic interactions. Under basic conditions, quisinostatis expected to possess a negative charge due to deprotonation of thehydroxamic acid group, suggesting an ionic mediated loading mechanism.Since the pH was only altered after NP formation, the ionization couldeither enable quisinostat retention within the core of the solid NPand/or increase the stability of quisinostat at the water-polymerinterface. To test whether quisinostat could be associating with thesurface of the NP (as opposed to the core), blank (no drug) NPs in theprimary emulsion were generated and quisinostat was added directly tothe evaporation phase under basic conditions (pH 10). This formulationcondition nearly doubled the effective drug incorporation over our priorattempts, achieving a quisinostat loading of 9.3% (QNP-4). Furtherincreases to the mass of quisinostat added to the aqueous phase, from 5mg to 7.5 or 10 mg, did not result in increased loading (QNP-6 andQNP-7) even at a higher pH (pH 11, QNP-9), supporting a saturableassociation of drug with the surface of the NP. Formulations at a pH 7or pH 9, while following an identical post-loading procedure, NP loadingdropped to 2.7% (QNP-5) and 5.3% (QNP-8), respectively. When the organicphase (DCM) was pre-evaporated prior to addition of quisinostat, with orwithout pH change, NP loading dropped to <3% (data not shown). Thus, thehighest effective loading of quisinostat (QNP-4) requires thedeprotonation of quisinostat at a pH above 10 and can be achieved afterNPs are formed but only in the presence of organic solvent. The increasein quisinostat loading as pH increases up to pH 10 with no increase seenat pH 11 supports an ionic association with the full ionization ofquisinostat occurring between pH 9 and 10.

One experimental concern is whether the loading measured in theseexperiments could reflect drug precipitates instead of NP-associateddrug. There are three pieces of evidence that contradict thispossibility. First, the optical quality of the emulsion ischaracteristic of ultra-small polymeric nanoparticles, possessing atranslucent/blue hue that is not observed when drug precipitates [32].Second, TEM characterization does not show drug precipitates (FIG. 2).Third, when PLA-PEG was excluded but post-loading fabrication conditionsotherwise maintained, only 10 μg of quisinostat was recovered.

Each NP formulation was also characterized by DLS to measure size andzeta potential. BNPs formed by our standard technique (neutral pHevaporation phase) possessed an average diameter of 96 nm and a zetapotential of −13 mV (Table 1). Alterations to the evaporation phase pHdid not significantly alter the biophysical properties of BNPs (data notshown). The presence of quisinostat resulted in NPs with a slightly moreneutral surface charge compared to NPs lacking quisinostat, but theamount of quisinostat loaded did not significantly affect the surfacecharge across QNP formulations. In contrast, the measured NP diameterspositively correlated with quisinostat loading, with the averagediameter increasing to 129 nm for the formulation with the highestloading (FIG. 3). This phenomenon is consistent with previous reportsshowing increased NP diameter when drugs are loaded onto the surface ofpolymeric NPs [33, 34]. These observations further support the drugloading measured represents NP-associated quisinostat, as opposed toprecipitated drug.

Quisinostat release from QNPs or as free drug at 37° C. in PBS wasmeasured by absorbance after 1, 2, 4, 6, 24, 32, and 48 h. Freequisinostat was completely released from the dialysis cassette by 4 h,whereas only 50% of quisinostat was released from NPs after 6 h, andcomplete NP release was achieved by 48 h (FIG. 4). The fast rate ofrelease from PLA-PEG NPs is in contrast to the 5 days of sustainedrelease previously reported for quisinostat encapsulated within the coreof PLGA-lipid hybrid NPs [22]. A rapid burst release supports surfaceloading of quisinostat [34, 35], and the subsequent phase of sustainedrelease is presumably due to electrostatic interactions with theparticle, which have previously been demonstrated to enable thesustained release of proteins from PLGA NPs, even in absence ofencapsulation [36]. It remains to be determined whether quisinostatresides within the hydrated PEG layer or is within the PLA polymer phaseand merely close to the surface. It is not immediately clear that theburst release is a problem for quisinostat drug delivery, since NPstypically distribute and clear over similar time frames to the releasekinetics observed here [37, 38].

The data demonstrate that the pH of the aqueous phase is a major forcedriving quisinostat loading into or onto PLA-PEG NPs formed by emulsion,and suggest that the mechanism is charge-mediated. A likely possibilityis that the deprotonation of quisinostat under basic conditionsincreases NP loading due to electrostatic interactions. Presumably, thepresence of the organic solvent is required to achieve this because itenhances overall solubility of the drug to enable this interaction.Previous works have described the loading of drugs and proteins onto thesurface of inorganic [39, 40] and polymeric [34-36] NPs. These effectshave been reported to be a function of charge interactions, [34-37] andtheir pH-dependency supports ionization as a primary mechanism [34, 36,39, 40]. Additionally, a charge-dependent loading of proteins onto thesurface of PLGA has been demonstrated in a post-fabrication scheme [36].However, to our knowledge, similar approaches have not yet beendemonstrated for loading small molecules on PLA-PEG, and have also notbeen reported for HDACis.

QNP activity and efficacy To test whether quisinostat potency ismaintained after NP loading, growth inhibition produced by free versusNP quisinostat in vitro was evaluated in GL261 cultures. Both free andNP-loaded quisinostat effectively inhibited the growth of GL261 cellswith IC50 values of 24 and 30 nM, respectively (FIG. 5). No significantchanges in quisinostat potency due to the NP loading process were found,and the low nanomolar IC50 is consistent with reported quisinostat IC50values against other glioblastoma cell lines [19].

Multiple investigators have identified HDAC inhibitors as drugs ofinterest for treating cancer, including GBM [1, 8, 17, 41-43]. While invitro results have been promising, little success has been observed invivo [20, 44, 45]. As a monotherapy, quisinostat and other HDIs haveshown the greatest in vivo efficacy against hematological cancers [17,19, 46]. Against solid tumors, HDIs are most commonly utilized as acombination therapy to achieve efficacy [6, 20, 22, 47]. Although themechanism for the in vivo failure of quisinostat or other HDIs as amonotherapy is unknown, it has been suggested that poor delivery may bea factor. NPs have the potential to improve in vivo efficacy ofsystemically administered agents through a variety of mechanisms,including improved solubility (enabling a higher dose to be delivered),enhanced permeation and retention (EPR) in leaky tumor vasculature,and/or alteration to pharmacokinetic profile of free drug. For example,in previous work, a NP encapsulation strategy was utilized to deliverthe otherwise ineffective drug camptothecin (CPT) to intracranial GBM[21]. CPT is a potent drug in cell culture but is very poorly watersoluble, inactivated at physiological pH, and cleared rapidly followingsystemic administration. Encapsulation of CPT withinpoly(lactic-co-glycolic acid) (PLGA) NPs improved drug tolerabilitydramatically, which produced a robust slowing of tumor growth andprolongation of survival in mice bearing intracranial tumors. Based onthis previous work, it was predicted that NP encapsulation would providea similar benefit to the action of quisinostat.

Prior works using hydroxy-propyl-β-cyclodextrin and/or mannitol tosolubilize quisinostat for injection report the maximum tolerated doseto be in the range of 35-70 mg/kg/week when administered by IP or SQinjection [18, 19, 46]. In these studies, mice did not show significantweight loss at QNP doses up to 100 mg quisinostat/kg/week IV (FIG. 6),suggesting an improvement in quisinostat tolerability after NPencapsulation. Quisinostat has previously shown efficacy againstsubcutaneous GBM xenografts [19], which confirms quisinostatdemonstrates expected activity against GBM but does not address deliverybarriers related to orthotopic tumors. Treatment of orthotopic GBM issignificantly hindered by the blood-brain barrier (BBB), which presentsboth active and passive barriers to restrict the entry of chemotherapies[48, 49]. Nearly all drugs of interest for GBM fail to achieve adequatetumor concentrations at a safe dose [50]. Thus, the inability ofsubcutaneous tumors to recapitulate these unique drug deliverychallenges makes intracranial GBM models necessary for evaluatingtreatment efficacy.

To test whether free quisinostat could treat an orthotopic tumor,intracranial GL261-LucNeo tumors were induced in 10 C57BL/6 albino miceand treated with either saline or free quisinostat (n=5/group) by IPinjection on days 11, 12, 18, and 19. Free quisinostat failed to provideany treatment benefit with a tumor doubling time of 2.4 days for bothtreatment and a median survival of 22 and 19 days for saline andquisinostat, respectively (FIG. 7). In a separate cohort of 12 micebearing intracranial tumors, the subjects were divided into 3 treatmentgroups (saline, BNPs or QNPs) and treatments were administered IV bylateral tail vein injection on days 11, 12, 18, and 19. Tumor growth wasexponential in both saline and BNP treated mice with an average tumordoubling time of 2.3 and 2.2 days, respectively, while QNPssignificantly (p<0.05) slowed the tumor doubling time, to 3.4 days (FIG.8). This delay in tumor growth resulted in a significant increase inmedian survival to 27.5 days for QNP treated compared to 21 days forthose treated with BNPs (p=0.03) and tended to prolong survival comparedto the 21.5 days for saline treated mice (p=0.10). Although a modestimprovement in survival, these data show NP encapsulation of quisinostatcan improve its tolerability and efficacy over free drug to effectivelyslow intracranial GBM growth as a monotherapy.

This disclosure presents a novel pH driven approach for achieving highquisinostat loading of PLA-PEG NPs, ˜9% (w/w), after NP formation. Incontrast to the typical approach of reducing drug solubility in theaqueous phase to drive partitioning of drug into the NP core, the datashow that quisinostat loading increases as its aqueous solubilityincreases, which likely is due to a charge-mediated association of drugwith the nanoparticle surface. QNPs produced by these methodseffectively release drug over 48 h and possess equivalent activity tofree drug in vitro. Additionally, QNPs were found to robustly sloworthotopic GL261 tumor growth and prolong survival compared to controltreated mice. These data support a novel mechanism for loading NPs withquisinostat and further the development of HDACis for the treatment oforthotopic glioblastoma.

It should be understood from the foregoing that, while particularembodiments have been illustrated and described, various modificationscan be made thereto without departing from the spirit and scope of theinvention as will be apparent to those skilled in the art. Such changesand modifications are within the scope and teachings of this disclosureas defined in the claims appended hereto.

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What is claimed is:
 1. A method of fabricating a therapeuticnanoparticle, comprising: a. preparing an aqueous phase; b. adjustingthe pH of the aqueous phase c. mixing an organic phase with the aqueousphase, wherein the organic phase comprises an organic solvent and ananoparticle comprising an amphiphilic polymer d. partially removing theorganic solvent to drive nanoparticle formation; e. after step (d),adding a water-insoluble biologically active ingredient, the activeingredient comprising an ionizable group; and f. removing the remainingorganic solvent.
 2. The method of claim 2, wherein the active ingredienthas a higher water solubility in the adjusted pH.
 3. The method of claim1, wherein the active ingredient is at least 70% ionized in the aqueousphase, the active ingredient and the nanoparticle electrostaticallyinteract.
 4. The method of claim 1, wherein the active ingredient is aweak acid, and the pH of the aqueous phase is between 8 and
 14. 5. Themethod of claim 1, wherein the active ingredient is a weak base, and thepH of the aqueous phase is between 1 and
 7. 6. The method of claim 1,wherein the active ingredient comprises an ionizable group with apartition coefficient of log P>0.
 7. The method of claim 1, wherein theionizable group is selected from the group consisting of hydroxamic acidgroup, carboxyl group, hydroxyl group, sulfhydryl group, phenolic group,amino group, imidazole group, guanidinium group, sulphonamide group, andimide group.
 8. The method of claim 1, further comprising dissolving theactive ingredient in a solvent selected from the group consisting ofdimethyl sulfoxide (DMSO), acetonitrile, and acetone.
 9. The method ofclaim 1, wherein the aqueous phase comprises a surfactant, a stabilizer,or both; and the surfactant or the stabilizer is selected from the groupconsisting of sodium cholate, sodium dodecyl sulfate, poloxamer, Tweens,vitamin E, tocopheryl polyethylene glycol succinate (TPGS), ethyleneglycol, glycerol, and polyvinyl alcohol (PVA).
 10. The method of claim1, wherein the organic solvent comprises a water-immiscible solventselected from the group consisting of dichloromethane (DCM), chloroform,carbon tetrachloride, dichloroethane, diethyl ether, ethyl acetate, andtoluene.
 11. The method of claim 1, wherein the organic solventcomprises a water-miscible solvent selected from the group consisting ofacetaldehyde, acetic acid, acetone, acetonitrile, cyclohexane,dimethylformamide, dioxane, ethanol, heptane, hexane, methanol, formicacid, ethylamine, dimethyl sulfoxide, pentane, propanol, pyridine, andtetrahydrofuran.
 12. The method of claim 1, wherein the nanoparticle isprepared by emulsification; and the method further comprises: a. forminga pre-emulsion organic phase comprising the amphiphilic polymer and theorganic solvent; b. combining the pre-emulsion organic phase with apre-emulsion aqueous phase to form a pre-emulsion mixture; and c.emulsifying the pre-emulsion mixture to form an emulsion.
 13. The methodof claim 1, wherein the amphiphilic polymer is selected from the groupconsisting of poly(lactic acid)-poly(ethylene glycol) (PLA-PEG),poly(lactic-co-glycolic acid)-poly(ethylene glycol),poly(lactic-co-glycolic acid)-d-α-tocopheryl polyethylene glycolsuccinate, poly(lactic-co-glycolic acid)-ethylene oxide fumarate,poly(glycolic acid)-poly(ethylene glycol),polycaprolactone-poly(ethylene glycol), and a combination thereof. 14.The method of claim 13, wherein the amphiphilic polymer comprisesPLA-PEG having a weight averaged molecular weight of 2,000 to 60,000daltons.
 15. The method of claim 1, wherein the therapeutic nanoparticlehaving at least 4%, at least 6%, or at least 9% of the active ingredient(% w/w).
 16. The method of claim 1, wherein active ingredient comprisesa histone deacetylase inhibitor selected from the group consisting ofvorinostat (suberoylanilide hydromaxic acid, SAHA), istodax, belinostat,apicidin, suberoyl bis-hydroxamic acid (SBHA), scriptaid, sodiumbutyrate, trichostatin A, entinostat, Panobinostat, mocetinostat,romidepsin, tubastatin A, givinostat, dacinostat, quisinostat,pracinostat, droxinostat, abexinostat, ricolinostat, tacedinaline,tubacin, resminostat, citarinostat, santacruzamate, nexturastat A,tasquinimod, parthenolide, and any pharmaceutically acceptable saltsthereof.
 17. The method of claim 1, wherein the hydrodynamic diameter ofthe therapeutic nanoparticle is between 20-300 nm.
 18. The method ofclaim 1, wherein the zeta potential of the therapeutic nanoparticle isbetween −35 and +10 mV.
 19. The method of claim 1, wherein the activeingredient is at least 30% partially loaded onto the surface of thepolymeric nanoparticle.
 20. The method of claim 1, wherein thetherapeutic nanoparticle further comprises a second biologically activeingredient.